Methods and compositions for high throughput identification of protein/nucleic acid binding pairs

ABSTRACT

Methods and compositions for high-throughput identification of protein/nucleic acid binding pairs are provided. In the subject methods, a nucleic acid probe array, e.g., a molecular beacon probe array, is contacted with a target nucleic acid population to produce a hybridized array. The resultant hybridized array is then contacted with a population of proteins to produce a protein bound array. Any resultant array surface bound target nucleic acid/protein complexes are then detected to identify protein/nucleic acid binding pairs. In certain embodiments, the protein and/or nucleic acid members of the identified protein/nucleic acid binding pairs are further characterized. Also provided are systems and kits for use in practicing the subject methods. The subject invention finds use in a variety of different applications.

FIELD OF THE INVENTION

The field of this invention is molecular biology, particularlyprotein/nucleic acid binding interactions and protocols for theidentification thereof.

BACKGROUND OF THE INVENTION

Identification of protein-nucleic acid interactions is paramount inunderstanding the underlying molecular mechanisms in cellular processessuch as replication, transcription, and signaling. One importantcomponent in the characterization of DNA/RNA binding proteins is theanalysis of sequence specific interactions using “footprinting”techniques, in which the sequence of the protein binding domain of anucleic acid is identified.

One footprinting protocol that finds use is based on ligation mediatedpolymerase chain reaction (LMPCR) (Mueller, P. R and Wold, B. (1989)Science 246: 780-786). Reagents that are commonly employed in thisprotocol include DNasel, DMS (dimethylsulfate) and UV light. In thesefootprinting protocols, a given nucleic acid, typically of knownsequence, is screened for the presence of protein binding sequences bycontacting the nucleic acid with one or more test nucleic acid bindingproteins. Specific sequences along the nucleic acid that are bound tothe protein(s) are protected from nucleophilic attack or cross-linkingby the reagents, thus creating a “footprint” across this region(s) inthe nucleic acid. The protected region is then identified by firstcleaving the DNA at the lesion, and annealing a gene specific primer tothe region of interest. This primer is extended using a processive DNAPolymerase to the cleavage site, creating a blunt end. A unidirectionallinker (staggered) is then attached to the blunt ended molecule usingDNA ligase. The 3′ end of the longer strand of the linker is ligated tothe 5′ end of the genomic DNA. The shorter strand of the linker lacks a5′ phosphate and therefore is not ligated to the extension product. Asecond gene specific primer and a linker specific primer are annealed tothis product, which is now a suitable substrate for a PCR reaction. Onlymolecules that have both sequences (primer 2 sequence and linkersequence) are amplified. A third gene specific primer (labeled) is thenused to sequence the products that can subsequently be visualized on asequencing gel. In this manner, the protein binding sequence of thenucleic acid is identified.

Terminal Transferase dependent PCR (TDPCR) is a modified LMPCRmethodology that has been devised for studying protein-RNA interactions(Tornaletti, S, and Pfeifer, G (1995) J. Mol. Biol. 249: 714-728; Chen,H-H, et al. (2000) Nucl. Acid Res. 28: 1656-1664). It uses UV light asthe primary source of creating appropriate lesions (intra-strandpyrimidine dimer formation, primarily between thymidines) within theRNA, which inhibit progression of DNA polymerases.

Although LMPCR and TDPCR are very powerful techniques in mappingprotein-nucleic acid interaction or binding sites, they suffer fromseveral disadvantages that are summarized below. First, in studyingprotein-nucleic acid interactions using LMPCR/TDPCR, one needs to haveprior knowledge of the gene sequence (or transcript) in question inorder to be able to design appropriate gene specific primers foramplification. Second, the LMPCR/TDPCR protocols are labor intensive andoffer considerable challenges to those not well. versed in the art.Third, both LMPCR and TDPCR allow analysis of protein-nucleic acidinteractions at the nucleotide resolution by revealing the footprintthat the protein leaves behind on the nucleic acid. However, they arenot useful techniques in determining the underlying identity of theprotein(s) resulting in such a footprint. To identify the proteins perse, one has to resort to the use of monoclonal antibody protocols, whichsuffer from the drawback that a priori knowledge about the identity ofthe proteins is needed. Because of the above limitations, none of thecurrently employed techniques for identifying protein/nucleic acidbinding pairs can be adopted for high throughput mapping ofsite-specific protein binding sequences.

As such, there is a continued interest in the development of newprotocols for identifying protein/nucleic acid binding pairs, where thedevelopment of a protocol that could be adapted to a high throughputformat is of particular interest.

Relevant Literature

U.S. Patents of interest include: U.S. Pat. Nos. 5,925,517; 6,150,097;6,355,421. Also of interest is: Tyagi & Kramer, Nat Biotechnol (March1996) 14(3): 303-8.

SUMMARY OF THE INVENTION

Methods and compositions for identifying protein/nucleic acid bindingpairs are provided. In the subject methods, a nucleic acid probe arrayis first contacted with a target nucleic acid population to produce ahybridized array. The resultant hybridized array is then contacted witha population of proteins to produce a protein bound array.Protein/nucleic acid binding pairs-are then detected on the arraysurface. In certain embodiments, the protein and/or nucleic acid membersof the identified protein/nucleic acid binding pairs are furthercharacterized.

In many embodiments, the array employed is a molecular beacon arrayhaving a plurality of distinct molecular beacon probes all labeled withthe same first fluorescent label. In these embodiments, the molecularbeacon array is first contacted with a target nucleic acid population toproduce a hybridized array. The resultant hybridized array is thencontacted with a population of proteins all labeled with the same secondfluorescent label to produce a protein bound array. A feature of themethods of this embodiment is that the first and second fluorescentlabels make up a FRET pair. Any FRET generated signals from theresultant protein bound array are then detected from the protein boundarray to identify protein/nucleic acid binding pairs.

Also provided are systems and kits for use in practicing the subjectmethods. The subject invention finds use in a variety of differentapplications.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a view of a representative molecular beacon probe of themolecular beacon arrays employed in certain embodiments of the subjectinvention.

FIG. 2 provides an illustration of the hybridization of a target nucleicacid to a molecular beacon probe and the consequent conformationalchange of the molecular beacon probe to provide for a detectable signal.

FIG. 3 provides an illustration of a protein bound to a target nucleicacid of a molecular beacon array, where the label of the protein and thelabel of the molecular beacon are in a FRET relationship.

FIG. 4 provides an illustration of the effect of distance on the FRETrelationship that can be established by the subject methods.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and compositions for high-throughput identification ofprotein/nucleic acid binding pairs are provided. In the subject methods,a nucleic acid probe array, e.g., a molecular beacon probe array, iscontacted with a target nucleic acid population to produce a hybridizedarray. The resultant hybridized array is then contacted with apopulation of proteins to produce a protein bound array. Any resultantarray surface bound target nucleic acid/protein complexes are thendetected to identify protein/nucleic acid binding pairs. In certainembodiments, the protein and/or nucleic acid members of the identifiedprotein/nucleic acid binding pairs are further characterized. Alsoprovided are systems and kits for use in practicing the subject methods.The subject invention finds use in a variety of different applications.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing various invention components that aredescribed in the publications, which might be used in connection withthe presently described invention.

As summarized above, the subject invention provides methods andcompositions for the high-throughput identification of protein/nucleicacid binding pairs. In further describing the subject invention, themethods will be described first in greater detail, followed by a reviewof the systems and kits provided by the invention for practicing thesubject methods.

Methods

As summarized above, the subject invention provides methods for thehigh-throughput identification of protein/nucleic acid binding pairs.More specifically, the subject invention provides methods of identifyingprotein/nucleic acid binding pairs that exist in a union of a first setof one or more nucleic acids and a second set of one or more proteins.The nucleic acid member of the identified binding pairs may be RNA,e.g., cRNA, mRNA etc., or DNA, e.g., single stranded or double strandedDNA. As such, RNA/protein binding pairs and DNA/protein binding pairsmay be identified by using the subject invention. The sets of nucleicacids and proteins that are screened or assayed according to the subjectmethods may be obtained from a variety of sources, including naturallyoccurring or synthetic sources. In addition, the sources of the proteinsand nucleic acids that make up the assayed sets may be the same ordifferent.

In practicing the subject methods, a nucleic acid probe array isemployed to assay the union of a set of nucleic acids and proteins forthe presence of protein/nucleic acid: binding pairs in the union. Toassay the union of the nucleic acid and protein sets, the nucleic acidprobe array is first contacted with a population of target nucleicacids, i.e., the set of target nucleic acids, to produce a hybridizedarray. The resultant hybridized array is then contacted with apopulation of labeled proteins, i.e., the set of proteins, to produce aprotein bound array. The resultant protein bound array is then assayedto detect any resultant surface bound labeled protein molecules in orderto detect protein/nucleic acid binding pairs that exist in the union ofthe assayed nucleic acid and protein sets. Depending on the particularembodiment, the labeling protocol employed to detect the surface boundprotein/nucleic acid complex may vary. Representative labeling protocolsinclude those that employ directly detectable labels and those thatemploy indirectly detectable labels, where the latter of which arecharacterized by having two or more signal producing system members thatwork in concert to produce a detectable signal. Examples of directlydetectable labels include isotopic labels, fluorescent labels, FETlabeling systems, including FRET labeling systems, etc. Examples ofindirectly detectable labels include those found in enzymatic signalproducing systems, e.g., chemillumninescent systems, etc.

Because of the ease of use and detection of fluorescent labels, in manyembodiments employed the labels employed are directly detectablefluorescent labels. Fluorescent labeling systems of interest include FETlabeling systems, where energy transfer between donor and acceptormoieties occurs, where the acceptor may be a second fluorescer, e.g., asis present in FRET systems, or may be a quencher moiety. In certainembodiments of particular interest, a FRET labeling system is employed,where at least two of three main assay components (i.e., the array boundprobe nucleic acids, the target nucleic acids, and the proteins) havelabels that form a FRET pair, and in certain embodiments all three ofthese components have labels that form a FRET pair.

In many embodiments of particular interest, the array of probe nucleicacids that is employed is one that is a molecular beacon array. Sincethese embodiments are of particular interest, the invention will now befurther described in terms of these embodiments.

In practicing the subject methods of these embodiments of particularinterest, a molecular beacon array is employed to assay the union of aset of nucleic acids and proteins for the presence of protein/nucleicacid binding pairs in the union. To assay the union of the nucleic acidand protein sets, the molecular beacon array is first contacted with apopulation of target nucleic acids, i.e., the set of target nucleicacids, to produce a hybridized array. The resultant hybridized array isthen contacted with a population of fluorescently labeled proteins,i.e., the set of proteins, to produce a protein bound array. Theresultant protein bound array is then assayed for any resultant FRETgenerated signals, which signals are then detected in order to detectprotein/nucleic acid binding pairs that exist in the union of theassayed nucleic acid and protein sets.

Molecular Beacon Array

As such, the first step in the subject methods is to provide a molecularbeacon array for use in the subject methods. The molecular beacon arrayis a composition of matter that includes a substrate that displays atleast one molecular beacon probe immobilized on a surface thereof, wherethe arrays employed in the subject invention typically include aplurality of distinct molecular beacon probes immobilized on a surfaceof a substrate, where each member of the plurality differs in terms ofprobe sequence, as described in greater detail below.

The molecular beacon probes of the subject arrays are conformationallylabeled probe structures that generate a different fluorescent signaldepending on whether or not they are hybridized to a target nucleicacid. In other words, the molecular beacon probes are probes thatgenerate a first fluorescent signal, e.g., a quenched signal,undetectable signal, when not hybridized to a target nucleic acid and asecond fluorescent signal, e.g., an unquenched fluorescent signal, whenhybridized to a target nucleic acid. While in principle anyconformational probe that functions as described above may be employed,in many embodiments the probes have a molecular beacon structure.

Molecular beacon conformational probe structures are known to those ofskill in the art and reviewed in, among other places, U.S. Pat. Nos.5,925,517; 6,150,097 and 6,355,421 (the disclosures of which are hereinincorporated by reference); as well as Tyagi & Kramer, Nat Biotechnol(March 1996) 14(3):303-8. Molecular beacons are single stranded nucleicacid or nucleic acid mimetic (e.g., PNA) probes that form a stem-loopstructure. A fluorophore, i.e., first fluorescent label, and quencherare linked to opposite ends of the molecule. Fluorescence is quenchedwhen the probe is in the stem-loop conformation. However, when the probesequence in the loop anneals to a complementary nucleic acid targetsequence, the duplex formed overcomes the shorter hairpin-stem so thatthe probe undergoes a conformational transition that separates thefluorophore and quencher, such that the signal generated by the firstfluorescent label upon excitation is no longer quenched. FIGS. 1 and 2provide a depiction of a representative molecular beacon probe in thetwo different conformations.

In the molecular beacon probes employed on the subject molecular beaconprobe arrays, the probe sequence of the stem-loop structure is designedto hybridize to at least a portion of a target nucleic acid sequence.The probe sequence length may be any convenient length. In manyembodiments, the length typically ranges from about 5 about 200residues, e.g., nt, PNA subunits, etc. Often, the probing nucleobasesequence will be 5 to 150 nt in length, e.g., 10 to 100 nt in length,such as 50, 60, 70 nt in length, etc.

Flanking either side of the probe sequences in the molecular beaconprobes are arm segments. The arm segments are designed to anneal to eachother and thereby stabilize the interactions that fix the energytransfer of linked donor and acceptor moieties, i.e., first fluorescentlabel and quencher therefore, until the molecular beacon probehybridizes to the target sequence. The arm segments may be of differentlengths, but are typically the same length. The preferred length of thearm segments will depend on the stability desired for the interactions.However, the arm segments must not be so long that they prohibithybridization to the target sequence. Often, the arm segments are fromabout 2 to about 10 subunits in length and more often from about 2 toabout 5 subunits in length. In certain embodiments, both arm segmentsare external to the probing sequence.

Each molecular beacon probe is labeled such that the probe yields aquenched or unquenched fluorescent signal, depending on the conformationof the molecular beacon probe. The labels attached to the probescomprise a set of energy transfer moieties comprising at least oneenergy donor and at least one energy acceptor moiety. Typically, the setincludes a single donor moiety and a single acceptor moiety.Nevertheless, a set may contain more than one donor moiety and/or morethan; one acceptor moiety. The donor and acceptor moieties operate suchthat the acceptor-moiety accepts energy transferred from the donormoiety, resulting in quenching of the signal from the acceptor moiety.

In many embodiments, the donor moiety is a fluorophore. Representativefluorophores are derivatives of fluorescein, derivatives of bodipy,5-(2′-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), derivativesof rhodamine, cyanine dyes, e.g., Cy2, Cy3, Cy 3.5, Cy5, Cy5.5, texasred and its derivatives, etc. Though the previously listed fluorophoresmight also operate as acceptors, in certain embodiments the acceptormoiety is a quencher moiety, e.g., a non-fluorescent aromatic orheteroaromatic moiety, e.g., 4-((-4-(dimethylamino)phenyl)azo)benzoicacid (dabcyl), etc.

Transfer of energy from the donor, e.g., first fluorescent label, mayoccur through collision of the closely associated moieties of a set orthrough a nonradiative process such as fluorescence resonance energytransfer (FRET). For FRET to occur, transfer of energy between donor andacceptor moieties of a set requires that the moieties be close in space(e.g., less than about 100 Å, often less than about 80 Å) and that theemission spectrum of a donor(s) have substantial overlap with theabsorption spectrum of the acceptor(s). Alternatively, collisionmediated (radiationless) energy transfer may occur between very closelyassociated donor and acceptor moieties whether or not the emissionspectrum of a donor moiety(ies) has a substantial overlap with theabsorption spectrum of the acceptor moiety(ies). This process isreferred to as intramolecular collision since it is believed thatquenching is caused by the direct contact of the donor and acceptormoieties.

The molecular beacon probes are generally polymeric and may be nucleicacids, polymeric mimetics thereof, e.g., PNAs, or copolymers ofnucleotide and non nucleotide residues, e.g., block copolymers ofnucleic acids and nulceic acid mimetics, such as PNAs. The nature of themolecular beacon probes may vary, so long as that function as describedabove.

As indicated above, in many embodiments an array of the above-describedmolecular beacon probes is employed. The molecular beacon probe arraysinclude at least two distinct molecular beacon probes that differ fromeach other with respect to their probing sequence, and yet are labeledwith the same first fluorescent label, e.g., donor label, as describedabove. The molecular beacon probes of the array are immobilized on e.g.,covalently (such as cross-linked or directly synthesized throughphosphoramidite linkage chemistry) or non-covalently (such as throughbiotin/avidin binding pair) attached to, different and known locationson the substrate surface. The probes may be attached to the surfacedirectly, or through a suitable spacer group, as is known in the arrayart. Each distinct molecular beacon probe of the array is typicallypresent as a composition of multiple copies of the probe on thesubstrate surface, e.g., as a spot or feature on the surface of thesubstrate. The number of distinct probes, and hence spots or similarstructures, present on the array may vary, but is generally at least1000, and may be as high as 25,000 or higher. The spots of distinctprobes present on the array surface are generally present as a pattern,where the pattern may be in the form of organized rows and columns ofspots, e.g. a grid of spots, across the substrate surface, a series ofcurvilinear rows across the substrate surface, e.g. a series ofconcentric circles or semi-circles of spots, and the like. The densityof spots present on the array surface may vary, but will generally be atleast about 10 and usually at least about 100 spots/cm², where thedensity may be as high as 10⁶ or higher, and in certain embodiments willgenerally not exceed about 10⁵ spots/cm². A variety of different arrayconfigurations and formats, including choice of substrate material,organization of probes, dimensions, etc., are known and have beendeveloped, where any convenient configuration may be employed.Representative configurations of interest include, but are not limitedto, those described in U.S. Pat. Nos. 5 6,372,483; 6,355,421; 6,323,043;6,306,599; 6,242,266; 6,222,030; 6,221,653; 6,180,351; 6,171,797; and6,077,674; the disclosures of which are herein incorporated byreference.

In certain embodiments, two or more distinct probes on the array form aset of probes that all hybridize to the same target nucleic acid, wherethe probe sequences of the different members of the set each hybridizeto different domains or regions of the same target nucleic acid. Seee.g., FIG. 4, where two probes that hybridize to the same target nucleicacid at different locations are illustrated. In certain embodiments, thearrays include sets of molecular probes that span the entire length of atarget nucleic acid, such that the entire sequence of the target nucleicacid is represented among the different molecular-beacon probes of theset that all hybridize to that target nucleic acid—in other words a“tiled” set of molecular beacon probes is provided for a target nucleicacid. Such embodiments find use in applications where characterizationof the cognate sequence of an identified protein/DNA binding pair isdesired, as described more fully below.

Target Nucleic Acid Hybridization

The next step in the subject methods is to bind the solid support boundmolecular beacon probe(s), e.g., molecular beacon array, with one ormore target nucleic acids under hybridization conditions to produce ahybridized array. In the broadest sense, the target nucleic acid(s)contacted with the array in this step is any nucleic acid, which is tobe screened or assayed together with a protein set to identify whetherit is part of a protein/nucleic acid binding pair. As such, the length,chemical nature and source of the target nucleic acid(s) may varygreatly, depending on the particular protocol being performed. Thenucleic acids may be oligonucleotides, polynucleotides etc. The nucleicacid may be RNA, e.g., cRNA, mRNA, etc., or DNA, including either singlestranded or double stranded DNA, e.g., cDNA, etc.

In many embodiments, a plurality of distinct nucleic acids are contactedwith the molecular beacon array, e.g., 5 different, 50 different, 100different, 500 different, 1000 different, 10,000 different, etc.,nucleic acids of differing sequence.

The plurality of target nucleic acids that is contacted with themolecular beacon array may be generated using any convenient targetnucleic acid generation protocol, where representative target generationprotocols include both linear and geometric amplification protocols,where the generated target nucleic acids may be DNA, RNA etc. In manyprotocols known to those of skill in the art, an initial nucleic acidbiological source is employed, e.g., a cellular or tissue nuclearsource. Any convenient nucleic acid source may be employed.

A representative protocol of particular interest in certain embodimentsincludes the linear amplification protocol described in U.S. Pat. No.6,132,997, the disclosure of which is herein incorporated by reference.

In many embodiments, the protocol that is employed is one that generatesunlabeled target nucleic acids, as a label element on the target nucleicacid is not employed and could, potentially though not necessarily,interfere with the signal producing system that is employed. If,however, the target nucleic acid is labeled, it its labeled with amoiety that does not adversely affect the signal producing systememployed in the subject methods, as described in greater detail below.

Once generated, the population of target nucleic acids is contacted withthe molecular beacon array under hybridization conditions to produce ahybridized array. In many embodiments, the hybridization conditionsunder which contact of the array and the target nucleic acids takesplace are stringent hybridization conditions. The term “stringenthybridization conditions” as used herein refers to conditions that arecompatible to produce duplexes on an array surface between complementarybinding members, i.e., between probes and complementary targets in asample, e.g., duplexes of nucleic acid probes, such as DNA probes, andtheir corresponding nucleic acid targets that are present in the sample,e.g., their corresponding cRNA analytes present in the sample. Anexample of stringent hybridization conditions is hybridization at 37° C.or higher and 0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate).Another example of stringent hybridization conditions is incubation at42° C. in a solution: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10%dextran sulfate, followed by washing in 0.5×SSC with 0.01% SDS followedby another wash of 0.06×SSC at about 65° C. Stringent hybridizationconditions are hybridization conditions that are at least as stringentas the above representative conditions, where conditions are consideredto be at least as stringent if they are at least about 80% as stringent,typically at least about 90% as stringent as the above specificstringent conditions. Other stringent hybridization conditions are knownin the art and may also be employed, as appropriate.

Contact/binding of the target nucleic acid population with the molecularbeacon array as described above results in the production of ahybridized array. As such, duplex nucleic acid structures are producedat any location where a target nucleic acid has hybridized to the probesequence of a surface bound molecular beacon probe. At these locationsof the array, the hybridization of the target nucleic acid to the proberesults in a conformational change of the probe, as illustrated in FIG.2.

Optionally, following production of the target hybridized nucleic acidarray, the hybridized array may be scanned or read, e.g., usingconventional fluorescence detection techniques as described in greaterdetail below, to identify the target nucleic acids present in thecontacted target nucleic acid population.

Protein Binding

Following production of the hybridized molecular beacon array, and anysignal detection step, e.g., fluorescence scanning step (as mentionedabove and described in greater detail below), the hybridized array iscontacted with at least one labeled protein. A feature of the labeledprotein is that it includes a second fluorescent label which, togetherwith the first fluorescent label of the surface bound molecular beacon,produces or makes up a FRET pair. Two fluorescent labels are viewed asbeing a FRET pair for purposes of the present invention if, whenpositioned sufficiently close to each other (typically less than about100 Å, and usually less than about 80 Å), they participate influorescence resonance energy transfer, such that excitation of one ofthe labels gives rise to emission from the other of the two labels. Avariety of FRET pairs of fluorescent labels are known to those of skillin the art and may be employed. The energy donors of the pairs willgenerally be compounds which absorb in the range of about 300 to about800 nm, more usually in the range of about 450 to about 700 nm, and arecapable of transferring energy to an acceptor fluorophore, whichgenerally absorbs light of a wavelength 15 nm, more usually 20 nm orhigher, than the absorption wavelength of the donor. The acceptor willgenerally emit in the range of about 400 to about 900 nm. Fluorophoresof interest include, but are not limited to: fluorescein dyes (e.g.,5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM),2′,4′,1,4,-tetrachlorofluorescein (TET),2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX), and2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE)), cyanine dyessuch as Cy5 and Cy3, dansyl derivatives, rhodamine dyes (e.g.,tetramethyl-6-carboxyrhodamine (TAMRA), andtetrapropano-6-carboxyrhodamine (ROX)), DABSYL, DABCYL, anthraquinone,nitrothiazole, and nitroimidazole compounds, and the like. Fluorophoresof interest are further described in WO 01/42505 and WO 01/86001, aswell as the priority U.S. Applications of these documents, thedisclosures of the latter of which are herein incorporated by reference.

Any convenient protocol may be employed to produce the labeled protein,as described above. In certain embodiments, the protein of interest islabeled with functionalized label reagent that covalently bonds to theprotein and, in doing so, labels the protein. In these embodiments, theprotein is contacted with functionalized label under conditionssufficient for a functional moiety of the protein, e.g., an amine orhydroxyl group, to react with the corresponding functional moietypresent on the label to produce a covalent bond between the label andthe analyte. As such, functionalized labels employed in theseembodiments of the subject methods include a functional moiety and alabel moiety. The functional moiety of the functionalized labels mayvary greatly, and is chosen in view of the functional moiety present onthe protein to be labeled, e.g., amine groups on the protein. In otherwords, the functional moiety present on the functionalized label is onethat reacts with the functional moiety present on the protein to producea covalent bond between the protein and the label. Representativefunctional moieties that may be present on the label include: amino,sulfhydryl, sulfoxyl, aminosulfhydryl, azido, epoxide, isothiocyanate,isocyanate, anhydride, monochlorotriazine, dichlorotriazine, mono-ordihalogen substituted pyridine, mono- or disubstituted diazine,maleimide, aziridine, sulfonyl halide, acid halide, alkyl halide, arylhalide, alkylsulfonate, N-hydroxysuccinimide ester, imido ester,hydrazine, azidonitrophenyl, azide, 3-(2-pyridyl dithio)-propionamide,glyoxal, aldehyde, iodoacetyl., cyanomethyl ester, p-nitrophenyl ester,o-nitrophenyl ester, hydroxypyridine ester, carbonyl imidazole, and thelike.

In many embodiments, the hybridized array in this step in contacted witha population of different proteins, i.e., a protein set, where theproteins are all labeled with the same second fluorescent label. Bypopulation of different proteins is meant a plurality of proteins thatdiffer from each other in terms of amino acid sequence, where the numberof distinct or different proteins in the population is at least 2,usually at least 50, more usually at least 100, and often is muchgreater, e.g., at least about 500, at least about 1000, at least about2000, at least about 5000 etc.

In many embodiments, the population of labeled proteins is produced bycontacting an initial source of a plurality of different proteins withfunctionalized label, as described above. The initial source ofdifferent proteins may be any convenient source, e.g., a syntheticsource, a naturally occurring source, e.g., a cell lysate, tissuehomogenate, etc.

At least one fluorescently labeled protein, i.e., the protein set, asdescribed above, is contacted with the hybridized array underprotein/nucleic acid binding conditions sufficient to produce a proteinbound array. Contact may occur using any convenient protocol. As such, afluid sample that includes the at least one fluorescently labeledprotein may be applied to the substrate surface, flowed across thesubstrate surface, or the substrate surface may be immersed in the fluidsample, etc.

Binding/contact between the surface and sample including the at leastone labeled protein is maintained for a period of time sufficient forbinding between the protein and any recognized nucleic acid bindingsequences present on the substrate surface to occur. As such, thesubstrate surface and the sample are incubated for a period of time andunder conditions sufficient for binding between nucleic acids andproteins of a given protein/nucleic acid binding pair to occur. Thesample and substrate are typically incubated for a period of timeranging from about 5 min to 2 hours, usually from about 15 min to 2hours and more usually from about 30 min to 1 hour. The temperatureduring this incubation period generally ranges from about 0 to about 37°C. usually from about 15 to 30° C. and more usually from about 18 to 25°C. Where desired, the substrate and sample may be agitated duringincubation, e.g., by shaking, stirring, etc.

The above contacting/incubating steps result in the production of aprotein bound array, which includes one or more surface boundprotein/nucleic acid binding pairs, if such pairs exist in thecollection or union of target nucleic acid and labeled protein sets thatare assayed according to the subject methods. The surface boundprotein/nucleic acid binding pairs may have a structure as illustratedin FIG. 3.

FRET Signal Detection

Following production of the protein bound array, the surface of thearray is assayed for the presence of FRET generated signal. Anyconvenient protocol for detecting FRET generated signal on the surfacemay be employed. Typically, this step involves irradiating the surfacewith a wavelength suitable for absorption of one of the fluorescentlabels so that a FRET generated emission from the other of thefluorescent labels is produced, followed by detection of this FRETgenerated signal. Any convenient protocol for irradiating at the firstwavelength and detecting the FRET emitted signal may be employed. Assuch, reading of the array may be accomplished by illuminating the arrayand reading the location and intensity of resulting fluorescence at eachfeature of the array to detect any protein/nucleic acid bindingcomplexes on the surface of the array. For example, a scanner may beused for this purpose, which is similar to the AGILENT MICROARRAYSCANNER scanner available from Agilent Technologies, Palo Alto, Calif.Other suitable apparatus and methods are described in U.S. patentapplications: Ser. No. 09/846125 “Reading Multi-Featured Arrays” byDorsel et al.; and Ser. No. 09/430214 “Interrogating Multi-FeaturedArrays” by Dorsel et al. These references are incorporated herein byreference.

Any detected FRET generated signals are then attributed to the presenceof a protein/nucleic acid binding pair at the location of the surfacefrom which the signal is generated. In this manner, detection of a FRETgenerated signal on the surface of the array is employed to detect aprotein/nucleic acid binding pair on the surface of the array.

In certain embodiments, the hybridized array is contacted with twodistinct populations of labeled proteins, which are differentiallylabeled. By differentially labeled is meant that the two populations arelabeled with different fluorescent labels that are distinguishable fromeach other, e.g., upon excitation they emit at different maxima.Although the two populations are differentially labeled, the label ofthe first population and the label of the second population mustnonetheless form a FRET pair with the first fluorescent label of thesurface bound molecular beacon. In such embodiments, the two differentprotein populations are generally contacted/bound in known amountsrelative to each other with the array, such that the ratio of amounts offirst and second populations contacted/bound to the array is known. Incertain embodiments, substantially equimolar, including equimolar,amounts of the first and second protein populations are contacted/boundwith the array. Embodiments where two differentially labeled proteinpopulations are; bound with the hybridized array include applicationswhere the identified protein/nucleic acid binding pairs are to bequantitated, where protein populations are to be compared, e.g.,normal/control pairs; disease/normal pairs, etc.

Results from the reading may be raw results (such as fluorescenceintensity readings for each feature in one or more color channels) ormay be processed results such as obtained by rejecting a reading for afeature which is below a predetermined threshold and/or formingconclusions based on the pattern read from the array (such as whether ornot a particular target sequence may have been present in the sample).The results of the reading (processed or not) may be forwarded (such asby communication) to a remote location if desired, and received therefor further use (such as further processing). In certain embodiments,the subject methods include a step of transmitting data from at leastone of the detecting and deriving steps, as described above, to a remotelocation. By “remote location” is meant a location other than thelocation at which the array is present and hybridization and/or proteinbinding occurs. For example, a remote location could be another location(e.g. office, lab, etc.) in the same city, another location in adifferent city, another location in a different state, another locationin a different country, etc. As such, when one item is indicated asbeing “remote” from another, what is meant is that the two items are atleast in different buildings, and may be at least one mile, ten miles,or at least one hundred miles apart. “Communicating” information meanstransmitting the data representing that information as electricalsignals over a suitable communication channel (for example, a private orpublic network). “Forwarding” an item refers to any means of gettingthat item from one location to the next, whether by physicallytransporting that item or otherwise (where that is possible) andincludes, at least in the case of data, physically transporting a mediumcarrying the data or communicating the data. The data may be transmittedto the remote location for further evaluation and/or use. Any convenienttelecommunications means may be employed for transmitting the data,e.g., facsimile, modem, internet, etc.

Identification of Protein/Nucleic Acid Binding Pairs

As indicated above, the data generated upon reading of the array isemployed to identify protein/nucleic acid binding pairs that exist inthe union of the set of proteins and nucleic acids that are assayed withthe array according to the subject methods. More specifically, the data,i.e., FRET generated signal, is employed to identify protein/nucleicacid binding pairs that exist in the combined set of target nucleicacids and labeled proteins that are contacted with the molecular beaconarray during practice of the subject methods. For example, where thetarget nucleic acid population and the labeled protein populationcontacted with the molecular beacon array are obtained from the samecellular/tissue source, any observed FRET generated signals indicate thepresence of protein/nucleic acid binding pairs found in the cell/tissuesource. In other embodiments where the target nucleic acid and proteinpopulations are from different sources, FRET generated signals indicateprotein/nucleic acid binding pairs present in the union of the two setsfrom different sources. As such, the array is scanned for the presenceof FRET generated signals, where any observed signals indicate thepresence of a protein/nucleic acid binding pair and therefore can berelated to the presence of a protein/nucleic acid binding pair, i.e.,the presence of a protein/nucleic acid binding pair can be derived fromthe observed FRET generated signal.

Optional Additional Steps

Following identification of the any protein/nucleic acid binding pairs,the identified protein/nucleic acid binding pairs may be furtheranalyzed, e.g., to identify the nature of the protein member and/ornucleic acid member of the pair.

Protein Identification

The protein member of the protein/nucleic acid binding pair may befurther characterized/identified using a number of different protocols,including protocols known to those of skill in the art. Basically, anyconvenient protocol may be employed, where the protocol yieldsadditional information with regard to the nature/identity of the proteinmember of the identified protein/nucleic acid binding pair. Onerepresentative protein characterization protocol that may be employed isto produce an enzyme digest profile for the protein, where the proteinis then compared to a reference database of digest profiles to identifythe protein. For example, the protein member of the protein/nucleic acidbinding pair may be digested with trypsin to produce a trypsin digest,where the resultant fragments are analyzed by tandem MALDI-TOF/ESI massspectrometry to produce a searchable profile. Identification of theprotein is then done by comparing the resultant profile to a database ofreference profiles generated by a theoretical trypsin digest createdagainst all available protein sequences in a given protein sequencedatabase (for example, SWISS-PROT). Such a protocol is described in:Gygi, S. P et al. Nat. Biotech. (1999), Vol 17: 994-999, and Griffin, T.J. et al. Anal. Chem. (2001), Vol 73: 978-986. Other proteincharacterization protocols that may be employed include, but are notlimited to: yeast two-hybrid protocols, protein fragment complementationassay protocols; and the like.

Where desirable, larger amounts of the protein member of theprotein/nucleic acid binding pair may be obtained prior tocharacterization. Any convenient protocol for obtaining larger amountsof the to be characterized protein member may be employed. For example,one may be use the nucleic acid member of the pair to purify additionalprotein from the original source employed in the methods, as describedabove. For example, the nucleic acid member of the identifiedprotein/nucleic acid binding pair may be amplified to produce solidphase capturable nucleic acids, e.g., the nucleic acid may be amplifiedusing 5′ end biotinylated gene specific primers. These resultantcapturable nucleic acids can be employed to capture the protein memberof interest, e.g., by contacting the capturable nucleic acids with asource of the protein to be identified, e.g., the cellular/tissueextract or lysate, under protein/nucleic acid binding conditions. Theresultant complexes are then purified, e.g., by Streptavidin coatedbeads, to obtain purified amounts of the protein member for subsequentcharacterization. The above protein purification protocol is merelyrepresentative, as any convenient protocol may be employed. Otherprotocols of interest include, but are not limited to: Gygi, S. P et al.Nat. Biotech. (1999), Vol 17: 994-999; and the like.

Nucleic Acid Binding Sequence Identification

In certain embodiments, the binding sequence of the nucleic acid membermay be characterized. In certain embodiments, as described above, thearray employed in the subject methods includes a plurality of molecularbeacon probes that each hybridize to the same target nucleic acid, wherethe distinct probe members of the plurality differ from each other byhybridizing to different locations of the target nucleic acid to whichthey hybridize. In those embodiments where a plurality of such probesare present for each target nucleic acid, e.g., it includes a tiled setof probes for a given target nucleic acid, some members of the probe setwill give rise to a FRET signal and some will not, as illustrated inFIG. 4. By knowing the sequence of the target nucleic acid, as well asthe sequence of the probe regions of the molecular beacon probes that doand do not give rise to a FRET signal, one can readily approximate thesequence of the protein binding domain of the target nucleic acid whichis bound by the protein member of the protein/nucleic acid binding pair.

Utility

The subject methods of identifying protein/nucleic acid binding pairscan be used in a variety of different applications. Representativeapplications of interest include research applications, where thesubject invention is employed to identify and characterizeprotein/nucleic acid binding pairs. As such, one can employ the subjectinvention to rapidly identify and characterize RNA/protein bindingpairs, single-stranded DNA/protein binding pairs (where the proteinmembers may be involved in DNA replication, repair, recombination,etc.), double-stranded DNA/protein binding pairs (where the proteinmembers may be histones, transcription factors, methylases, polymerases,etc.), telomeric DNA/protein binding pairs, secondary structure (e.g.,Z-DNA, G-quartet DNA, triplex DNA, cruciforms, etc.) assuming nucleicacid/protein binding pairs, etc., in various research applications, suchas elucidation of biochemical pathways, e.g., cellular processes such asreplication, transcription, signaling, etc.

Systems

Also provided are systems for use in practicing the subject methods. Thesystems typically include at least the following components which areemployed in practicing the subject methods: (a) a molecular beaconarray; (b) protein labeling reagents, where the label of the labelingreagent and the label of the molecular beacon probes of the array makeup FRET pair; (c) target nucleic acid generation reagents; (d) afluorescent signal detector. Specifics regarding each of these elementsare provided above.

Kits

Also provided are kits for use in the subject invention. The kitstypically include a molecular beacon array and at least one proteinlabeling reagent where the labeling reagent includes a fluorescent labelthat is selected to make up a FRET pair with the fluorescent label onthe probes of the molecular beacon array, where FRET pairs are describedabove. In certain embodiments, the kits also include reagents necessaryfor generating the target nucleic acids, e.g., buffers, primers,polymerases, RNA isolation reagents, detergents, etc.

The various components of the kits may be present in one or morecontainers, each with one or more of the various reagents (sometimes inconcentrated form) utilized in the methods.

Finally, the kits may further include instructions for using the kitcomponents in the subject methods. The instructions may be printed on asubstrate, such as paper or plastic, etc. As such, the instructions maybe present in the kits as a package insert, in the labeling of thecontainer of the kit or components thereof (i.e., associated with thepackaging or sub-packaging) etc. In other embodiments, the instructionsare present as an electronic storage data file present on a suitablecomputer readable storage medium, e.g., CD-ROM, diskette, etc.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental

I. Preparation of Molecular Beacon Arrays-Molecular Beacon Arrays arePrepared by the Following Methods.

A. Deposition

In one method, the individual oligonucleotide sequences labeled with theappropriate molecular beacons (fluorophore donors and acceptors) areprepared by conventional DNA synthesis on solid support using knownmethods, such as, but not limited to, the phosphoramidite strategy onCPG. When an additional synthetic moiety is necessary to anchor thesequences to a solid support, it is attached to the oligonucleotidesequence during the chemical synthesis of the individual probe, eitherat the 5′ or 3′ end, or in the center of the sequence. Thoseindividually prepared probes are then deposited on solid supports, suchas glass, by pulse-jet printing or by mechanical methods involving thecontact between the solid support and a physical carrier (fiber optics,pins, etc.) to produce a molecular beacon array. Typically, the glasssurface is functionalized prior to deposition with a mono or multilayerof a coating reactive with a natural or synthetic moiety within theoligonucleotide being deposited. The glass surface is optionally treatedafter deposition to covalently fix the DNA molecules and/or toinactivate the coating reactive groups that were not used in theattachment of the DNA sequences.B. In Situ Synthesis

In another method, the molecular beacon probes are synthesized directlyon the solid support, such as glass, in a spatially controlled manner toachieve the formation of individual features to produce a molecularbeacon array. The synthesis is typically performed using thephosphoramidite synthetic methodology and the spatial control isachieved during the coupling step using pulse-jet printing technologiesto deposit the phosphoramidite reagents. Other steps of the DNAsynthesis cycle are performed in a flowcell without spatial control.Alternative methods may include the spatial control of the deblock steputilizing, for instance, light activation strategies using photolabileprotection groups or photogenerated acids and bases. The solid supportis typically functionalized with moieties reactive with the first DNAmonomer coupled to the surface, such as hydroxyls or amino groups. Atthe end of the DNA synthesis, protecting groups, such as of the basesand phosphate groups, are removed under alkaline conditions which do notcleave the DNA probes from the surface.

The molecular beacon probes sequences are typically anchored to thesurface by their 3′ end, although existing chemistry permits theattachment at any location along the sequence, including the 5′ end. Aspacer is typically used between the molecular beacon DNA sequence andthe attachment point of the probe with the solid support. Typicalspacers include polyethylene glycol phosphates and polynuleotides ofnatural, such as T, and synthetic, such as such abasic, nucleic acidmonomers. The fluorophore acceptor is typically placed between thespacer and the DNA sequence, and the fluorophore donor is typicallyplaced at the other extremity of the DNA sequence. The DNA sequence ofthe first 8 bases and of the last 8 bases are chosen to be complementaryto each other to form, in the absence of DNA target, thethermodynamically favored stem loop. The DNA sequence between theflanking stem sequences can be any sequence of natural and modifiednucleic acids monomer necessary to capture the nuclei acid targets.

II. Preparation of Nucleic Acid Targets

Target nucleic acids are prepared using already established protocols(e.g., single stranded and double stranded c-DNA prepared by reversetranscription of mRNA.) Total RNA is prepared by precipitation ofnucleic acids from cellular extracts and subsequent DNAsel digestion.cRNA is prepared by the method of U.S. Pat. No. 6,132,997, thedisclosure of which is herein incorporated by reference.

III. Hybridization of Probe:Target Pair

Mix 2-5 μg of unlabeled target nucleic acid (see section II above) in atotal volume of 300 μl of hybridization buffer (e.g., from Agilent, PaloAlto, Calif.) in a hybridization chamber. Incubate the chamber in a 60°C. rotisserie oven with mixing for a period of 12-17 hrs. Dismantle thearray from the chamber at room temperature in a low stringency buffersuch as 6×SSPE (containing 0.005% sodium lauryl sarcosine) and wash thearray in the same buffer composition for 1 minute. Transfer the array toa fresh solution of high stringency buffer such as 0.06×SSPE and washfurther for 30 seconds to dissociate non-specifically bound targetmolecules.

IV. Preparation of Labeled Proteins

This protocol will apply for a library of expressed His tagged proteins.Clone a specific cDNA library (from tissue samples being compared) in anappropriate expression vector cassette containing an in-frame histidinetag. Transfect cells (mammalian, bacterial, insect etc . . . ) with thislibrary allowing for expression of the individual His-Tagged proteins.Concentrate cells by centrifugation and resuspend in 1 ml of Lysisbuffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0). Addlysozyme to a final concentration of 1 mg/ml. Incubate on ice for 30minutes and sonicate mixture to lyse cells. Centrifuge lysate at10,000×g for 30 minutes at 4° C. and collect the supernatant.Equilibrate Ni—NTA spin column with 600 μl of lysis buffer bycentrifuging for ˜2 minutes at 700×g. Load an equivalent volume oflysate containing the His-tagged proteins onto this pre-equilibratedcolumn and centrifuge for 2 minutes at 700×g. Wash the column twice with600 μl of wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH8.0) and centrifuge for 2 minutes at 700×g. Elute the His-taggedproteins twice with 200 μl of elution buffer (50 mM NaH₂PO₄, 300 mMNaCl, 250 mM imidazole, pH 8.0) and collect the eluate.

Label the proteins using the appropriate “protein labeling kit”available from Molecular Probes, Inc. and its associated protocol (e.g.,Fluorescein-EX Protein Labeling Kit”).

V. Binding Labeled Proteins to Hybridized Array

Perform labeled protein binding experiments by titrating, for eachmolecular beacon array, a specific concentration of purified labeledproteins (2-10 μg) under physiological buffer conditions (50 mM NaH₂PO₄,100 mM NaCl, 1 mM MgCl₂, 1 mM ZnCl₂, 1 mM CaCl₂, and proteaseinhibitors, pH 7.0-8.0) for a period of 1 hour at 37° C. Gently wash theprotein bound array with the binding buffer (50 mM NaH₂PO₄, 100 mM NaCl,1 mM MgCl₂, 1 mM ZnCl₂, 1 mM CaCl₂, and protease inhibitors, pH 7.0-8.0)for 30 sec.

It is evident from the above results and discussion that the subjectinvention provides a number of advantages over the current nucleicacid/protein binding pair characterization protocols described in theBackground of the Invention Section, above. Unlike LMPCR/TDPCR wheresequence: information is required to footprint the protein-nucleic acidcontacts, the subject microarray based technology outlined in thisinvention has this information built-in (as the sequence of thetranscript/probe attached on the surface). In addition, unlikeLMPCR/TDPCR, this technology is technically less challenging and caneasily be practiced by those with moderate familiarity with microarrays,gene expression profiling and protein expression, purification, andlabeling. Furthermore, one of the major advantages of this invention isthat, unlike LMPCR/TDPCR, this technology is very high throughput andcan identify numerous different protein(s) that bind to differentfeatures (each feature representing a particular transcript). LMPCR andTDPCR are not at all amenable to high throughput analysis and requireseveral days for data processing. As such, the subject inventionrepresents a significant contribution to the art.

All publications and patent application cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1-29. (Cancelled)
 30. A method of identifying protein/nucleic acidbinding pairs, said method comprising: (a) contacting a molecular beaconarray comprising a plurality of distinct molecular beacon probes,wherein each distinct probe of said plurality comprises a differentprobe sequence and all of said probes of said plurality share a commonfirst fluorescent label, with a population of fluorescently labeledproteins to produce a protein bound array, where each member of saidpopulation of fluorescently labeled proteins is labeled with a secondfluorescent label that makes up a FRET pair with said first fluorescentlabel; and (b) detecting any FRET generated signals from said array toidentify protein/nucleic acid binding pairs on said array.
 31. Themethod according to claim 30, wherein said method further comprisescharacterizing the protein of a protein/nucleic acid binding pairidentified by said method.
 32. The method according to claim 30, whereinsaid method further comprises characterizing the protein bindingsequence of a nucleic acid of a protein/nucleic acid binding pairidentified by said method.
 33. The method according to claim 30, whereinsaid array is contacted with two differentially labeled proteinpopulations.
 34. The method according to claim 33, wherein said twodifferentially labeled protein populations make up a test/control pair.35. The method according to claim 33, wherein said two differentiallylabeled protein populations make up a normal/disease pair.
 36. A systemfor use in identifying protein/nucleic acid binding pairs, said systemcomprising: (a) a molecular beacon array comprising a plurality ofdistinct molecular beacon probes, wherein each distinct probe of saidplurality comprises a different probe sequence and all of said probes ofsaid plurality share a common first fluorescent label; (b) a labelingreagent for labeling a protein population with a second fluorescentlabel, wherein said first and second labels make up a FRET pair; and (c)a fluorescence detector device.
 37. The system according to claim 36,wherein said system includes two different labeling reagents forproducing two differentially labeled protein populations that are eachlabeled with a different second fluorescent labeled that makes up a FRETpair with said first fluorescent label.
 38. The system according toclaim 36, wherein said fluorescence detector device is a fluorescentscanner.
 39. The system according to claim 36, wherein said systemfurther comprises reagents necessary for identifying a protein componentof an identified protein/nucleic acid binding pair.
 40. A kit for use inidentifying protein/nucleic acid binding pairs, said kit comprising: (a)a molecular beacon array comprising a plurality of distinct molecularbeacon probes, wherein each distinct probe of said plurality comprises adifferent probe sequence and all of said probes of said plurality sharea common first fluorescent label; and (b) a labeling reagent forlabeling a protein population with a second fluorescent label, whereinsaid first and second labels make up a FRET pair.
 41. The kit accordingto claim 40, wherein said kit includes two different labeling reagentsfor producing two differentially labeled protein populations that areeach labeled with a different second fluorescent labeled that makes up aFRET pair with said first fluorescent label.
 42. The kit according toclaim 40, wherein said kit further comprises reagents necessary foridentifying a protein component of an identified protein/nucleic acidbinding pair.
 43. A substrate comprising a surface having at least oneprotein/nucleic acid binding pair immobilized thereon, wherein eachprotein/nucleic acid binding pair comprises: (a) a molecular beaconprobe comprising a first fluorescent label; and (b) a fluorescentlylabeled protein labeled with a second fluorescent label and bound tosaid probe, wherein said second fluorescent label and said firstfluorescent label make up a FRET pair.
 44. The substrate according toclaim 43, wherein said substrate comprises two or more differentprotein/probe binding pairs immobilized on said surface.
 45. The methodaccording to claim 30, wherein said method further comprises a datatransmission step in which a result from a reading of the array istransmitted from a first location to a second location.
 46. The methodaccording to claim 45, wherein said second location is a remotelocation.
 47. A method comprising receiving data representing a resultof a reading obtained by the method of claim
 30. 48. A method ofidentifying protein/nucleic acid binding pairs, said method comprising:(a) contacting a nucleic acid probe array comprising a plurality ofdistinct probe nucleic acids, wherein each distinct probe nucleic acidof said plurality comprises a different probe sequence, with apopulation of labeled proteins to produce a protein bound array; and (b)detecting any surface bound protein/target nucleic acid complexes toidentify protein/nucleic acid binding pairs on said array.
 49. Themethod according to claim 48, wherein said labeled proteins are labeledwith a first fluorescent label.
 50. The method according to claim 48,wherein said labeled proteins are labeled with an indirectly detectablelabel.
 51. The method according to claim 48, wherein said method furthercomprises contacting said array with a second population of labeledproteins that are distinguishably labeled from said first population oflabeled proteins.
 52. A method of identifying protein/nucleic acidbinding pairs, said method comprising: (a) contacting a molecular beaconarray comprising a plurality of distinct molecular beacon probes,wherein each distinct probe of said plurality comprises a differentprobe sequence and all of said probes of said plurality share a commonfirst fluorescent label, with at least one fluorescently labeled proteinto produce a protein bound array, where said at least one fluorescentlylabeled protein is labeled with a second fluorescent label that makes upa FRET pair with said first fluorescent label; and (b) detecting anyFRET generated signals from said array to identify protein/nucleic acidbinding pairs on said array.
 53. A method of identifying protein/nucleicacid binding pairs, said method comprising: (a) contacting a nucleicacid probe array comprising a plurality of distinct probe nucleic acids,wherein each distinct probe nucleic acid of said plurality comprises adifferent probe sequence, with at least one labeled protein to produce aprotein bound array; and (b) detecting any surface bound protein/targetnucleic acid complexes to identify protein/nucleic acid binding pairs onsaid array.